This project addresses the cellular signaling cascade in endocrine and neuroendocrine cells, and the interactions between plasma membrane electrical events and receptor-mediated intracellular signaling and secretion. Current emphasis is on native and recombinant nucleotide receptors (P2Rs) and nitric oxide (NO)-cGMP signaling pathways. Work on native P2Rs was done in anterior pituitary cells. Our results indicate that these cells secrete ATP and express G protein-coupled receptors (P2YRs) and cation-conducting channels (P2XRs). ATP and ADP, but not UTP and UDP, triggered calcium signaling in a majority of lactotrophs and prolactin (PRL) release in mixed pituitary cells, and their actions were abolished in the presence of apyrase, an ectonucleotidase. Transcripts for Gq-coupled calcium-mobilizing P2Y1R, P2Y2R, P2Y4R, and P2Y6R, as well as Gi-coupled P2Y12R, were identified in mixed anterior pituitary cells. The pharmacological profile of calcium mobilization-dependent signaling and PRL secretion indicated that P2Y1R mediates the stimulatory action of ATP and ADP. Pituitary cells also express P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R. Several lines of evidence suggested that P2X4R subtype provides a major pathway for calcium influx-dependent signaling and PRL secretion. Work on recombinant rat P2XR was focused on calcium signaling by these channels and the contributions of distinct receptor subdomains to the subtype-specific behavior. Recombinant P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to non-desensitizing): P2X3R > P2X2b+X4R > P2X2bR > P2X2a+X4R > P2X4R > P2X2aR > P2X7R. When expressed in GT1 cells, all wild type and chimeric P2XRs responded to agonist binding with global calcium signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of calcium signals was present independently of the rate of current-desensitization. The temporal characteristics of calcium signals were not affected by voltage-gated calcium influx and removal of extracellular sodium. Calcium signals reflected well the receptor-specific EC50s for ATP and the extracellular zinc and pH sensitivities of P2XRs. To tested the hypothesis that affinity of agonists for binding domain accounts for ligand-specific desensitization pattern, we generated chimeras using receptors with variable sensitivity to ATP in order: P2X4R > P2X2aR = P2X2bR >> P2X7R. Chimeras having the ectodomain Ile66-Tyr310 sequence of P2X2R and Val61-Phe313 sequence of P2X7R in the backbone of P2X4R were expressed, but were non-functioning channels. P2X2a+X4R and P2X2b+X4R chimeras having the Val66-Tyr315 ectodomain sequence of P2X4R in the backbones of P2X2aR and P2X2bR were functional and exhibited increased sensitivity to ligands compared to both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X2bR and P2X2aR, chimeric P2X2b+X4R desensitized more rapidly than P2X2a+X4R, and the rate of desensitization of P2X2a+X4R increased by substituting its Arg371-Pro376 intracellular C-terminal sequence with Glu376-Gly381 sequence of P2X4R. These results indicate the relevance of interaction between ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rates of receptor-desensitization, but does not affect the C-terminal-specific pattern of desensitization. The coupling between NO-cGMP signaling pathway and PRL release in pituitary lactotrophs has been established previously. However, the messenger that mediates the action of this signaling pathway on hormone secretion and the secretory mechanism affected, calcium-dependent or independent, have not been identified. Inhibition of constitutively expressed neuronal NO synthase decreased NO and cGMP levels and increased basal PRL release. The addition of a slowly releasable NO donor increased cGMP levels and inhibited basal PRL release in a time-dependent manner. Expression of inducible NO synthase also increased NO and cGMP levels and inhibited basal, depolarization-induced, and TRH-induced PRL release, whereas inhibition of this enzyme decreased NO and cGMP production and recovered PRL release. None of these treatments affected spontaneous and stimulated voltage-gated calcium influx. At basal NO levels, the addition of permeable cGMP analogs did not inhibit PRL secretion. At elevated NO levels, inhibition of cGMP production and facilitation of its degradation did not reverse inhibited PRL secretion. These experiments indicate that NO inhibits calcium-dependent PRL secretion in a cGMP-independent manner and downstream of voltage-gated calcium influx.